Background & objectives: The nature of the rickettsial antigens and the immune response generated by them, have been the subject of exhaustive research so that a suitable vaccine can be developed. Till date evaluations of Rickettsia rickettsii antigens that induce both humoral and cellular responses in animal models have only shown partial protection and short-term immunological memory. This study was aimed to evaluate the immune response induced by DNA plasmids generated from the OmpA and OmpB genes of R. rickettsii in peripheral blood mononuclear cells of rickettsial (sensitized) patients compared to healthy subjects.Methods: Plasmids OmpA-49, OmpB-15 and OmpB-24 were generated in the pVAX vector. Macrophages derived from the THP-1 cell line were transfected in vitro with the plasmids and were co-cultured with T-lymphocytes from sensitized subjects and healthy subjects to evaluate cell proliferation and cytokine production.Results: The OmpB-24 plasmid induced proliferative response in human lymphocytes, with production of IL-2, IFN-γ, IL-12p70, IL-6 and TNF-α, likely due to the presence of conserved epitopes among R. rickettsii, R. typhi and R. felis (differing from 1 to 3 amino acids) during the construction of the plasmids.Interpretation & conclusion: DNA sequences of rickettsial epitopes can be cloned into the pVAX vector. Constructed plasmids can generate a proliferative response and produce cytokines in vitro, in co-culture of transfected macrophages with sensitized human lymphocytes. Plasmid OmpB-24 proved to be the most immunogenic with respect to plasmids OmpA-49 and OmpB-15.

Rickettsia rickettsii, the causal agent of Rocky Mountain spotted fever (RMSF), is an obligate intracellular bacterium that resides mainly in ticks of the family Ixodidae[1]. It often generates self-limited clinical pictures, but may cause long-term sequelae, or can be fatal, if not treated[2]. Currently, there is no effective vaccine against rickettsial diseases and the development of suitable control strategies is important, since there are no commercial methods available for its diagnosis, particularly in vulnerable communities that are away from the specialized health services, and it also has clinical symptoms similar to other pathologies, such as dengue, that might delay the treatment[3].

Vaccines using crude antigens or inactivated microorganisms were partially protective and had adverse reactions[4],[5],[6],[7]. The generation of DNA vaccines against the OmpA and OmpB rickettsial proteins have shown increased immunity in murine models infected with R. conorii and R. rickettsii[8]. Presently, the most promising DNA vaccine is based on R. rickettsii OmpA protein which stimulates a protective immune response in mice with a lethal dose of R. conorii. In addition, the production of IFN-γ by T-lymphocytes suggests stimulation of a cellular immune response[9].

Recombinant antigens from genotypically conserved OmpB sequences to all rickettsial species have been used to evaluate their protection against lethal infection with R. conorii in C3H/HeN mice. The recombination of the surface protein Adr2 with OmpB has also been evaluated. The results showed that each protein individually induced a significant specific response, supporting the role of OmpB. However, evaluation of OmpA and OmpB proteins in murine models might be a limiting factor when compared with human models. The use of transgenic mice in human leukocyte antigen type-1 (HLA-I) and HLA-II, as well as humanized mice capable of expressing various human HLA, have been reported as feasible strategies with great human implication[10],[11],[12].

Because of the intracellular nature of R. rickettsii, the experimental strategy to follow is to identify T-lymphocytes, that specifically recognize the epitope processed and expressed in HLA-I molecules. Naturally activated T-lymphocytes (first exposure) can be reactivated in vitro (second exposure), and their immune activity can be compared with that of the lymphocytes from healthy subjects. This strategy has been evaluated in lymphocyte cultures of patients sensitized with R. typhi or R. prowazekii with lymphoblastic cells infected with the same rickettsial species, confirming the effect of cytotoxic T-lymphocytes and cross-immunity[13],[14].

Ex vivo trials, using macrophages or dendritic cells as antigen presenting cells (APC) in co-culture with sensitized lymphocytes, generate high expectations with high predictive value and greater human profile than any preclinical strategy[15],[16],[17]. These trials supplemented with in silico studies support the potential of APC-based antigen strategies in search of potential vaccine candidates, as documented for R. prowazekii and R. typhi[18]. Although OmpA and OmpB have been proposed as protective antigens, there is no evidence that these proteins effectively prevent rickettsial diseases[19]. The objective of the present study was to evaluate the immunostimulating effect of DNA plasmids generated from OmpA and OmpB genes of R. rickettsii in mononuclear cells of patients with positive diagnosis of Rickettsia.

Material & Methods

Plasmid DNA constructs

OmpA-49 (1.7 Kb), OmpB-15 (1.2 Kb) and OmpB-24 (1.7 Kb) fragments containing the majority of the open reading frame for OmpB and OmpA of R. rickettsii, were amplified and cloned by using site-specific PCR primers.

The alignment temperature and MgCl2 concentration were as follows: 66°C and 2.5 mM for OmpA-49, 60°C and 2.5 mM for OmpB-15, and 60°C and 3.5 mM for OmpB-24. The reaction mixture included 100 ng of DNA, 0.1 UI of Taq DNA polymerase, 20 μM of each deoxyribonucleotide (dATP, dCTP, dGTP, and dTTP), and 0.4 μM of each primer, in a final reaction volume of 25 μl. Amplification conditions were as follows: 30 cycles at 95°C for 1 min and 72°C for 2 min of final extension. The amplification products (10 μl) were separated and identified by electrophoresis in an 8% polyacrylamide gel stained with ethidium bromide (0.5 mg/ml), and visualized in an ultraviolet light (UV) Transilluminator. The fragments were ligated to the mammalian expression vector pVAX (Invitrogen, Carlsbad, California) and transformed into competent E. coli DH5-a cells according to the manufacturer’s instructions. Transformants bearing plasmids containing rickettsial DNA fragments were identified by sequencing. Orientation and frame relative to the cytomegalovirus promoter, were confirmed by DNA sequence analysis with ABI PRISM® 310 (Applied Biosystem, USA) sequencer at the Institute of Biotechnology, Cuernavaca City, Mexico. All plasmid constructs were maintained in the E. coli transformants under kanamycin selection in ZMY505 medium, and large-scale concentrated plasmid preparations (250 μg/ml)of these constructs were generated using HiPure Plasmid Filter Maxiprep kit (Invitrogen, CA, USA) according to the manufacturer’s instructions[20].

Plasmids expressed in macrophages: To determine the plasmid expression macrophages transfected were harvested and treated with TRIzol reagent for RNA extraction, following the manufacturer’s recommendations. The purified RNA was resuspended in DEPC water, quantified by spectrophotometry, and stored at –70 °C until use. The cDNA was obtained from RNA by reverse transcriptase reaction using the ImProm II kit (Promega, USA). PCR was performed using the conditions described for OmpA-49, OmpB-15, and OmpB-24 sequences.

OmpA-49, OmpB-15, and OmpB-24 detected by indirect immunofluorescence: Polyclonal antibodies against the OmpA-49, OmpB-15 and OmpB-24, were generated in BALB/c mice. Groups of mice were immunized with plasmids expressing the sequences at intervals of 15 days. One group of mice was inoculated with the plasmid pVAX without insert as a negative control. At Day 45, mice were bled by cardiac puncture to obtain serum. Polyclonal sera with titres 1: 64 were incubated with transfected macrophages for 30 min at 37 °C on culture plates. The antimouse IgG antibody conjugated to fluorescein (FITC) was used to detect the reaction according to the conditions of the manufacturer (Sigma, San Luis, Missouri, USA), and visualized in a fluorescence microscope. Performance was calculated by the ratio of fluorescence (fluorescent cells/cells in visible light × 100), averaging five fields. Serum obtained from mice inoculated with empty pVAX was used as a negative control.

Study population

A total of 14 patients and 14 healthy subjects as controls, matched for age (not older than 65 yr) and gender, were included in the study. Patients were clinical cases with positive serology and PCR to rickettsia, detected as part of community service at the Laboratory of Emerging Diseases and Re-emerging of the Regional Research Center “Dr Hideyo Noguchi” of the Autonomous University of Yucatan, Mexico. Blood samples from patients and healthy subjects were obtained under prior informed consent. Healthy subjects or patients with any acute infection or newly vaccinated were excluded.

Ethical approval

The study was approved by the Ethics Committee of Agustín O’Horán Hospital. Confidentiality of participants was strictly maintained.

Serological diagnosis of patients: Patients serum samples, diluted in 2% PBS/BSA (1: 64 titer), were incubated for 1 h at 37 °C on slides containing Vero cells infected with R. rickettsii and R. typhi[21]. Subsequently, FITC-conjugated anti-IgM (Sigma F5384) or IgG (Sigma F6380) secondary antibody diluted 1: 100 in PBS/2% BSA was added, incubating at 37 °C for 1 h. At the end of the incubation the slides were washed with PBS, allowed to dry and the solution Airvol (Polyvinyl alcohol) (Sigma Aldrich P8136) was added. The samples were observed on a fluorescence microscope (Nikon Eclipse, TS-100F, UK). A negative serum sample and pathogens related to Rickettsia were used as a negative control.

Characterization of rickettsial species involved in clinical cases by PCR/RFLP: The diagnosis of rickettsia was established by PCR for Rickettsia 17 kDa and citrate synthase (gltA) genes as described previously[9],[22]. Positive controls were DNA ofR. felis, R. rickettsii, R. akari, and R. typhi, and a reaction without DNA was used as a negative control. The DNA of patients and controls were handled separately to avoid contamination.

Rickettsia rickettsii was identified as the causal agent by restriction fragment length polymorphism (RFLP) analysis of the amplified fragment of gltA (382 bp) and 17-kDa gene (434 bp) by using AluI as described previously[22],[23], and by comparing the DNA sequences of the gltA and 17-kDa gene PCR amplicons using BLAST software of the National Centre for Biotechnology Information (Bethesda, MD, USA)[24],[25].

Proliferation assays

Mononuclear peripheral blood cells (PBMC) were obtained from heparinized venous blood (15 ml) of patients and healthy subjects by centrifugation with Ficoll gradient (Nycoprep, Axis Shield). PBMCs were collected, washed and cultured in RPMI-1640 fetal serum free to promote adhesion of monocytes. Non-adherent cells (lymphocytes) were recovered and co-cultured for 72 h with transfected macrophages (1.5 × 105 per well) at 2 : 1 in RPMI-1640 fetal serum free with antibiotics, for 72 h at 37°C in 5% CO2 and 95% humidity. Triplicate assays were performed using macrophages transfected with empty vector pVAX, and untransfected cells (SC) as negative controls, and stimulated macrophages with BCG vaccine as a positive control.

Cell proliferation was measured by colorimetric reaction (reduction Methyl Tetrazolium salts) using an ELISA reader at a range of wavelength 500 to 600 nm. A non-parametric Wilcoxon test for paired data and student’s t-test were performed to determine significant differences (p < 0.05) between the proliferation of lymphocytes in the patient group and the group of healthy control subjects using three plasmids.

Detection of cytokines

Levels of IL-2 (DRG Diagnostics, Marburg, Germany), IFN-γ (DRG Diagnostics, Marburg, Germany), IL-12p70 (DRG Diagnostics, Marburg, Germany), TNF-α (R & D Systems, Minneapolis, Minnesota, USA) and IL-6 (Invitrogen, CA, USA) were determined in culture supernatants by ELISA. Samples were analysed in duplicate and concentrations calculated from the absorbance values were recorded using the standard curve constructed for each cytokine according to the supplier’s directions. Index absorbance, lymphoproliferation and statistical analyses were performed in SPSS (IBM) programme and the Wilcoxon test for paired data.

In silico analysis of OmpA and OmpB sequences of R. rickettsii

Rickettsia rickettsii sequence (Gene Bank accession number cp000848.1) was analysed for the presence of at least one peptide with affinity to HLA-I alleles A0201, A24, B3501 and B3901 (unpublished data). Rickettsial species sequences and bioinformatics were analysed using the Clustal W package, and BioEdit software.

Results

Cloning of rickettsial OmpA-49, OmpB-24 and OmpB-15 gene fragments

The amplified OmpA-49, OmpB-24 and OmpB-15 (PCR products) were successfully cloned into pVAX vector and used to transfect macrophages.

OmpA and OmpB sequences expressed in macrophages

Transcription of the R. rickettsii OmpA and OmpB sequences in transfected macrophages: Plasmids containing rickettsial OmpA-49, OmpB-24 and OmpB-15 fragments were purified and successfully transfected with cationic lipid transfection method into macrophages. The plasmid vector pVAX contains human cytomegalo virus (CMV) immediate-early promoter/enhancer that permits efficient, high-level expression of the exogenous protein. Amplification of the fragments after 48 h transfection was verified by RT-PCR. The results showed that the transfection was carried out efficiently since plasmids were capable of transfecting cells under a liposome system and able to generate the transcripts [Figure 1].

Study population: The analysis of clinic characteristics of both the groups [14 subjects with positive diagnosis of Rickettsia (8 with R. felis, 5 with R. typhi, 1 with R. rickettsii) and 14 healthy subjects] are summarized in [Table 1].

Rickettsial fragments presented by macrophages transfected to sensitized lymphocytes: To determine, whether lymphocytes from patients previously infected with Rickettsia show memory in response to cloned proteins, they were co-cultured with transfected macrophages and the proliferative response was assessed by MTT colorimetric test, at 72 h. Data showed significant differences in lymphoproliferative response to OmpA-49 and OmpB-24 in patients relative to controls. OmpB-15 showed no significant difference between the study groups [Figure 3]a. The lymphoproliferative response in patients who became sick due to R. felis, R. typhi and R. rickettsii, using plasmid OmpA-49, showed a significant increase (p = 0.0001) in two patients with R. typhi and one with R. felis, with respect to the control subjects [Figure 3]b. No significant differences (p = 0.6698) were found in the lymphoproliferation induced by macrophages transfected with plasmid OmpB-15 between patients analysed. Furthermore, analysis showed a significant increase (p = 0.0001) in proliferation of lymphocytes from 10 patients (4 to 6 for R. typhi and R. felis) employing the OmpB-24 plasmid [Figure 3]b.

Cytokines produced by lymphocytes: IL-2 was found increased in 11 supernatants of co-transfected cultures with OmpA-49, three with OmpB-15 and in nine supernatants with OmpB-24. Significant differences (p < 0.05) were observed between the levels of IL-2 from patients and control subjects with plasmid OmpA-49. Although no significant difference was observed between patients and controls by using the plasmid OmpB-24, the group of patients produced higher levels of IL-2. This was because the standard deviation and scattering of data was large, which prevents demonstration of statistical significance between the different data sets [Figure 4]a.

IFN-γ was found increased in four supernatants of co-transfected cultures with OmpA-49, six with OmpB-15, and in four with OmpB-24. Significant differences were observed in the levels of INF-γ between the patient group and the control group [Figure 4]b.

Cytokines produced by macrophages: IL-12p70 was increased in three supernatants with the three plasmids. The patients group showed statistically significant differences in levels of this cytokine by employing transfection with plasmid OmpB-24 indicating that this plasmid favours its production [Figure 4]c. TNF-α values above the mean were obtained in 11 supernatants with OmpA-49, 10 with OmpB-15, and in 12 with OmpB-24. Results showed highly significant differences between patients and controls with the three plasmids [Figure 4]d. IL-6 was found to be increased in 11 supernatants with OmpA-49, 13 with OmpB-15, and in 11 with OmpB-24. Significant differences were obtained between patients and controls with the three plasmids [Figure 4]e. Macrophages with empty vector pVAX and untransfected cells did not stimulate lymphoproliferation or cytokine secretion.

In sílico analysis of OmpA and OmpB from R. rickettsii str. ‘Sheila Smith’

The OmpA protein contains 14 epitopes, of which only one can be coupled to three HLA-I alleles (promiscuity) while the rest are promiscuous for two HLA-I alleles. The OmpB protein contains five epitopes of which one exhibit promiscuity for four alleles (position 24) when analysed in 10 different Rickettsia species; one peptide is promiscuous for three alleles and the three remaining are promiscuous in alleles. The expression and coupling affinity to HLA-I for all peptides was ≥ 70. Analysis in the BLAST search engine indicated that peptide does not present homology with human sequences. Highly conserved epitopes were identified in the OmpA and OmpB sequences rationalizing the cross-proliferation results obtained in the lymphocytes of patients who became diseased by R. thypi, R. felis and R. rickettsii. The OmpB sequence presented a non-conserved epitope (TLIEKLKDL) which could be used for the development of a possible specific species vaccine [Table 2].

Three vaccine candidates were generated from OmpA and OmpB membrane proteins: OmpA-49; OmpB-15 and OmpB-24. The immunogenic activity of these antigens has been reported in murine models for their lymphoproliferative capacity and production of INF-γ[9],[19]; however, it was demonstrated, for the first time, that OmpA-49 and OmpB-24 are capable of inducing T-lymphocyte proliferation and cytokine production using an expression system based on cationic liposomes in macrophages co-cultured with human lymphocytes. This expression system allowed us to immunodetect the HLA-I/rickettsial peptide complex in the macrophages membrane, and to evaluate its stimulating activity on human lymphocytes.

Differences in lymphoproliferative response observed in sensitized patients with R. typhi (2/6) and R. felis (7/14) might be due to the diversity of HLA alleles in patients or by changes on amino acids in Rickettsia strains. Bearing in mind the differences in the binding capacity between each peptide and the HLA alleles, it is possible that some will attach with greater affinity, remaining may anchor for longer duration to maintain the stimulus. This has been evaluated by in vitro assays of epitope-MHC binding[26],[27]. On the other hand, variations on amino acids in Rickettsia strains could be related with the severity of each infection. This corresponds with what has been described in infected individuals with R. felis in endemic areas of Brazil and Mexico[28],[29].

The low proliferation observed in the cells of the infected patient with R. rickettsii[Figure 3]b may be due to other factors such as the presence and time of circulation of cells with immunological memory, since the patient presented the infection symptoms eight years ago. CD4+ and CD8+ memory cells have been documented to remain 8–12 and 8–15 yr, respectively. It has been reported that, according to the persistence period of some microorganisms such as T. cruzi, CD8+ T-lymphocytes can be identified phenotypically in early (CD27+, CD28+), intermediate (CD27+, CD28– or CD27–, CD28+) and late stages of differentiation (CD27–, CD28–). The protective immunity could be conferred by the first ones with high proliferation capacity, and the last ones could be associated with chronic infections, as they show signs of senescence, and their re-stimulation and persistent activation could develop a type of energy or antigen tolerance[30],[31]. It is possible that the low proliferative response observed in the R. rickettsii or R. felis infected patient [Figure 3]b is given by late differentiation in CD8+ T-cells, however, this need to be evaluated.

Cytokines detected in culture supernatants in response to OmpA-49, OmpB-15 and OmpB-24 DNA plasmids, were those produced by T-lymphocytes (IL-2, IFN-γ) and macrophages (TNF-α, IL-12 and IL-6). The results suggest that the plasmids orient towards a Th1 response to the second exposure with the rickettsial antigen. IL-2 levels have been reported to increase in the first 24 h of culture, and decreases when soluble receptor (sIL-2R) increases[32],[33]. A significant difference (p < 0.05) was observed in IL-2 levels between patients and controls at 72 h of OmpA-49 culture [Figure 4]a. IL-2, produced mainly by NK and CD4+ Th1 lymphocytes, play an anti-rickettsial role since these induce the proliferation of CD8+ T-lymphocytes. The data suggest that IL-2 levels induced by OmpA-49 can activate CD8+ T-lymphocytes and favour the cytotoxic response against rickettsial antigens; nonetheless, further studies are needed to identify these cells and evaluate their activities against the antigenic stimulus.

The presence of IFN-γ, the product of the antigenic activation of CD8+ lymphocytes, Th1 and NK, induces a cellular immune response. Its production at the endothelium level favouring, the destruction of cells infected with Rickettsia has been reported[3]. Significant differences were not found in the levels of INF-γ, between patients and controls, after stimulation with the plasmids [Figure 4]b. It has been reported that IFN-γ synthesis begins at 8 h of culture in rickettsia-activated macrophages, peaking at 24 h. Its synthesis is regulated by the presence of IL-10 and IL-12, suggesting that continuous exposure of these cytokines might decrease the expression of IFN-γ RNA, and suppress the cellular immunity mechanism, leading to fatal cases in R. rickettsii infections[34]. Data of our experiment matched with the low cellular response reported during episodes of lymphocytic proliferation and cellular damage[34].

It was also observed that IL-12p70 levels in cultures of some patients decreased in response to stimulation with plasmids. However, a significant increase was observed in OmpB-24 in patients compared to the control group. Stimulation of PBMC with soluble R. conorii antigens has been reported to decrease IL-12p70 due to the presence of IFN-γ[35]. This could explain the low levels detected [Figure 4]c. However, IL-12p70 levels in response to OmpB-24 seemed to favour the overexpression of IFN-γ as observed in the study.

The three plasmids induced a significant increase in TNF-α and IL-6 in the cultures of patients compared to the controls, evidencing the presence of sensitized cells. It has been reported that these cytokines increase in the presence of rickettsial bacteria, focusing on their chemotactic and apoptosis effect at the sites of infection. On the other hand, overexpression of TNF-α has been described in murine and in vitro models in both sensitized and non-sensitized cells with rickettsial antigens[35],[36],[37],[38]. The levels of TNF-α detected in studied patients, in response to plasmids support their role in the inflammatory response and in the proliferation of sensitized cells. Regarding IL-6, increased production between 48 and 96 h of infection with R. typhi and R. parkeri has been observed in murine models, and it is suggested that it takes part in the purification of rickettsiae[39],[40],[41]. In the present case, OmpB-24 could have favoured the elimination of Rickettsia, which correlated with IL-6 levels and elevated lymphoproliferation rates observed in sensitized patients [Figure 4]e.

On the other hand, the significant levels of IL-6 and TNF-α observed in the patients, compared with healthy subjects, might also be due to the recognition of endogenous pathogen-associated molecular patterns (PAMPs). The presence of rickettsial genetic material in the plasmids could be an endogenous PAMP recognized by intracellular receptors such as DNA-dependent activators of IRFs (IFN regulatory factors) that activate the signalling pathway to induce the type-I IFN synthesis and proinflammatory cytokines in transfected macrophages[42].

The cross-responses obtained with lymphocytes from patients sensitized with R. typhi and R. felis may be due to the presence of conserved epitopes in the sequences of the analysed species and the ability of the antigenic epitopes to couple with the HLA-I alleles. Taking into account that each individual possesses six HLA-I alleles, the presence of peptides in the cloned OmpA and OmpB regions can be coupled with up to four alleles: HLA-A0201, HLA-A24, HLA-B3501 or HLA-B3901. In this regard, the allele A-0201 showed high affinity and longer dissociation time. This variant has an important global distribution, and several studies have considered it in the evaluation of cellular response during the selection of immunodominant peptides associated with CD8+ lymphocytes in microorganisms like T. cruzi[26],[43]. Thus, the design of a vaccine from these antigens would increase the population coverage and impact especially on vulnerable endemic areas[44]. This is important since the application of vaccines may not be restricted to specific populations where HLA allelic diversity plays a crucial role, and should favour its application to a larger population[45],[46],[47]. It is also encouraging that the cross-response to R. typhi and R. felis, possibly due to the presence of conserved peptides, favours the observed proliferation rates, generating higher expectations of cross-protection against similar species.

Conclusion

OmpA-49 and OmpB-24 rickettsial DNA plasmids generated and expressed in macrophages are capable of inducing the proliferation of sensitized human lymphocytes. The production of IL-2, IFN-γ, IL-6 and TNF-α from OmpB-24 showed a greater immune-stimulating effect than the others two plasmids, independent of the Rickettsia species involved in the infectious process, likely due to the presence of conserved antigenic epitopes and their binding capacity with the HLA-I alleles.

Conflict of interest

The authors declare to not have any conflict of interest.

Acknowledgements

This work was supported by the Mexican National Council for Science and Technology, grant CONACYT FOSISS 261885. The authors are grateful to Karina López and Raul Tello for their technical support.